Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.     

Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy

Small proteins provide convenient models for computational studies of protein folding and stability, which are usually compared with experimental data. Until recently, the unfolding of Trp-cage was considered to be a two-state process. However, no direct experimental evidence for this has been presented, and in some cases, the contrary has been suggested. To elucidate a detailed unfolding mechanism, we studied the thermodynamics of unfolding of Trp-cage by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The observation that at low temperatures only approximately 90-95% of Trp-cage exists in the native conformation presented an analytical challenge. Nevertheless, it was found that the DSC and CD data can be fitted simultaneously to the same set of thermodynamic parameters. The major uncertainty in such a global fit is the heat capacity change upon unfolding, DeltaCp. This can be circumvented by obtaining DeltaCp directly from the difference between heat capacity functions of the native and unfolded states. Using such an analysis it is shown that Trp-cage unfolding can be represented by a two-state model with the following thermodynamic parameters: Tm = 43.9 +/- 0.8 degrees C, DeltaH(Tm) = 56 +/- 2 kJ/mol, DeltaCp = 0.3 +/- 0.1 kJ/(mol.K). Using these thermodynamic parameters it is estimated that Trp-cage is marginally stable at 25 degrees C, DeltaG(25 degrees C) = 3.2 +/- 0.2 kJ/mol, which is only 30% more than the thermal fluctuation energy at this temperature.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.     

Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima.

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.     

Differential scanning calorimetry of the unfolding of myosin subfragment 1, subfragment 2, and heavy meromyosin

The thermal unfolding of rabbit skeletal heavy meromyosin (HMM), myosin subfragment 1, and subfragment 2 has been studied by differential scanning calorimetry (DSC). Two distinct endotherms are observed in the DSC scan of heavy meromyosin. The first endotherm, with a Tm of 41 degrees C at pH 7.9 in 0.1 M KCl, is assigned to the unfolding of the subfragment 2 domain of HMM based on scans of isolated subfragment 2. The unfolding of the subfragment 2 domain is reversible both in the isolated form and in HMM. The unfolding of subfragment 2 in HMM can be fit as a single two-state transition with a delta Hvh and delta Hcal of 161 kcal/mol, indicating that subfragment 2 exists as a single domain in HMM. The unfolding of subfragment 2 is characterized by an extraordinarily large delta Cp of approximately 30,000 cal/(deg.mol). In the presence of nucleotides, the high-temperature HMM endotherm with a Tm of 48 degrees C shifts to higher temperature, indicating that this peak corresponds to the unfolding of the subfragment 1 domain. This assignment has been confirmed by comparison with isolated subfragment 1. The stabilizing effect of AMPPNP was significantly greater than that of ADP. The vanadate-trapped ADP species was slightly more stable than M.AMPPNP with a Tm at 58 degrees C. The unfolding of subfragment 1, both in the isolated form and in HMM, was irreversible. Only a single endotherm was noted in the DSC scans of the subfragment 1 domain of HMM and in freshly prepared subfragment 1 complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.     

Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry

alpha-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 99.10 kcal mol(-1), and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96+/-0.18 kcal mol(-1)K(-1). The stability of alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.     

A monofunctional and thermostable prephenate dehydratase from the archaeon Methanocaldococcus jannaschii

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.     

Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.

Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.     

Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Equilibrium unfolding mechanism of chicken muscle triose phosphate isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.     

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.     

Kinetic stability plays a dominant role in the denaturant-induced unfolding of Erythrina indica lectin

The urea-induced denaturation of dimeric Erythrina indica lectin (EIL) has been studied at pH 7.2 under equilibrium and kinetic conditions in the temperature range of 40-55 degrees C. The structure of EIL is largely unaffected in this temperature range in absence of denaturant, and also in 8 M urea after incubation for 24 h at ambient temperature. The equilibrium denaturation of EIL exhibits a monophasic unfolding transition from the native dimer to the unfolded monomer as monitored by fluorescence, far-UV CD, and size-exclusion FPLC. The thermodynamic parameters determined for the two-state unfolding equilibrium show that the free energy of unfolding (DeltaGu, aq) remains practically same between 40 and 55 degrees C, with a value of 11.8 +/- 0.6 kcal mol(-1) (monomer units). The unfolding kinetics of EIL describes a single exponential decay pattern, and the apparent rate constants determined at different temperatures indicate that the rate of the unfolding reaction increases several fold with increase in temperature. The presence of probe like external metal ions (Mn2+, Ca2+) does not influence the unfolding reaction thermodynamically or kinetically; however, the presence of EDTA affects only kinetics. The present results suggest that the ability of EIL to preserve the structural integrity against the highly denaturing conditions is linked primarily to its kinetic stability, and the synergic action of heat and denaturant is involved in the unfolding of the protein.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.

Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.     

Increased thermal stability of proteins in the presence of naturally occurring osmolytes.

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential assembly of the tropomyosin heterodimer: equilibrium studies

Thermal unfolding/refolding studies of the three tropomyosin dimers, alpha alpha, alpha beta, and beta beta, from chicken gizzard muscle were performed to explain the preferential assembly of alpha- and beta-tropomyosin subunits into heterodimers, alpha beta [Lehrer, S. S., & Qian, Y. (1989) J. Biol. Chem. 265, 1134]. Circular dichroism measurements showed that all three dimers unfolded in cooperative reversible transitions with T1/2 = 40.0 degrees C and delta H degrees = 162 kcal/mol for alpha alpha and with T1/2 = 42.6 degrees C and delta H degree = 98 kcal/mol for beta beta at 0.4-0.5 microM concentrations. Fluorescence measurements on pyrenyliodoacetamide-labeled tropomyosin showed that (i) excimer fluorescence decreases in parallel with unfolding of homodimers, (ii) at physiological temperature, heterodimers are formed from micromolar mixtures of homodimers over a period of minutes, and (iii) heterodimers unfold/refold with temperature without appreciable formation of homodimers. To understand the preferential formation of alpha beta, we calculated the concentrations of all species present as a function of temperature for equal total amounts of alpha and beta, using the measured thermodynamic constants of the unfolding/dissociation equilibria for alpha alpha and beta beta. Values for delta H degrees = 225 kcal/mol and T1/2 = 43 degrees C for unfolding of alpha beta at 0.5 microM concentration were obtained from the best fit of the calculations to the measured helical content vs temperature of alpha beta.(ABSTRACT TRUNCATED AT 250 WORDS)